Quantitative analysis of a biological sample of unknown quantity

ABSTRACT

Disclosed is a method for testing a modified specimen such as a dried blood spot, plasma or serum specimen, for an analyte of interest, such as cholesterol. In accordance with the disclosed subject matter, the level of the analyte of interest in the medium from which the modified specimen was obtained (e.g., from a patient&#39;s blood) is determined based on the level of an analyte in a solution formed from the modified specimen and on the level of at least one normalizing analyte. The analyte and normalizing analyte each may be an ion, compound, biochemical entity, or property of the specimen. Also disclosed are a fluid collector and a fluid collection device.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/706,321, filed Nov. 12, 2003, which was acontinuation-in-part of U.S. patent application Ser. No. 10/421,086,filed Apr. 23, 2003, which claims priority to prior Application No.60/374,629 filed Apr. 23, 2002. All prior applications are herebyincorporated by reference in their entireties.

TECHNICAL FIELD OF THE INVENTION

The invention is in the field of testing, in particular quantitativetesting, and in preferred embodiments medical testing. In highlypreferred embodiments, the invention is directed towards the testing ofbody fluid specimens, in particular blood or serum specimens.

BACKGROUND OF THE INVENTION

Modern medical and wellness practices increasingly make use ofself-administered tests and self-collection of test specimens. Forinstance, U.S. Pat. Nos. 5,978,466; 6,014,438; 6,016,345; and 6,226,378,issued to Richard Quattrocchi and assigned to Home Access HealthCorporation of Hoffman Estates, Ill., all disclose a method ofanonymously testing for a human malady. In accordance with certainembodiments of the subject matter disclosed in the foregoing patents, apatient obtains a blood specimen, typically by pricking his or herfinger, and allows the blood to wick onto a blood spot card. After thecard has dried, the user then sends the blood spot card to a medicaltesting facility, where it is tested to determine whether the patient isafflicted with a specific malady. The user may contact the facilityanonymously to receive the test result.

The subject matter of the foregoing patents is usable in connection withtesting for the presence of human antibodies directed against viralantigens in the blood, for instance, in determining whether a patient isinfected with HIV (human immuno-deficiency virus) or with a hepatitisvirus. Another document, U.S. Pat. No. 5,435,970, issued to Mamenta etal. and assigned to Environmental Diagnostics, Inc. of Burlington, N.C.,discloses a device for separating blood cells from biological fluids,for instance, for separating serum from whole blood. The devicedisclosed in the '970 patent purports to enable the shipment and testingof a serum sample.

The blood spot and serum specimen cards known in the art are suitablefor use in the collection of specimens for qualitative testing, i.e.,testing for the presence or absence of a given compound in blood or agiven medical condition. Heretofore, however, such blood spot and serumcards have been somewhat unsatisfactory in the quantitative testing ofblood and serum specimens.

For instance, general wellness protocol indicates the measurements of apatient's total cholesterol value, which is the number of milligrams oftotal cholesterol in a deciliter of blood. The value is often used inconjunction with a full lipid profile, which provides levels oftriglycerides, HDL (high density lipoprotein) cholesterol, and LDL (lowdensity lipoprotein) cholesterol in a patient's blood. It can be verydifficult to gauge the amount of blood or serum that is present in theblood or serum spot card. Particularly when the blood or serum spot cardhas been self-prepared by a person without medical training, it isdifficult to know to certainty whether the spot card has been“underfilled” with less than the intended quantity of blood or serum or“overfilled” with more than the intended quantity. If the amount ofblood and serum varies by even a small amount over or under the expectedlevel, the usefulness of the quantitative test can be severelydiminished. For instance, it is generally thought that a person's totalcholesterol number should be under 200 mg/dl, with cholesterol numbersabove 240 mg/dl being considered high and with intermediate cholesterolnumber being deemed borderline. A 10% margin of error in a cholesteroldetermination of 220 mg/dl provides no information as to whether theperson's cholesterol level is low, intermediate, or high.

In recognition of these problems, the prior art has provided attempts toprovide a quantitative determination of analyte levels in a bloodspecimen. For instance, U.S. Pat. No. 6,040,135, issued to Steven Tyrelland assigned to Biosafe Laboratories, Inc., Chicago, Ill., purports todisclose a method for correcting for blood volume in a serum analytedetermination. The method that is purportedly disclosed by this documentis limited and is believed generally to be somewhat unsatisfactory.

The invention seeks to improve upon prior art testing methods, and toprovide a method for quantitative testing of modified specimens such asdried blood spot and dried serum specimens.

THE INVENTION

The invention provides multiple embodiments in the field of testing, inparticular medical testing. In accordance with the invention, a modifiedspecimen, preferably a dried blood fluid sample, such as a dried serumor dried whole blood specimen of unknown quantity, is eluted(re-solubilized) and then tested for an analyte. The level of analyte inthe blood from which the modified blood specimen was obtained isdetermined from the level of analyte in a solution formed from the bloodspecimen. A normalizing analyte, which in the preferred embodiment issodium ion, chloride ion, and/or osmolality, is measured and is used inconjunction with the solution level of analyte to determine the level ofanalyte in the blood from which the modified specimen was obtained. Theinvention is not limited to the field of medical testing but, to thecontrary, is useful in connection with other forms of testing. Theinvention further provides methods for preparing a database of testresults, for preparing a regression using a database of test results,and for providing test results to a user.

In alternative embodiments the invention further encompasses a fluidcollector that includes an absorbent substrate coated with a saccharide.A device that includes the collector (as described hereinbelow) also isencompassed by these embodiments.

Other features of preferred embodiments of the invention are set forthhereinbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flowchart representing steps in a method for calculating thelevel of an analyte in blood from which a blood specimen was obtained.

FIG. 2 is a flowchart representing steps in an alternative method forcalculating the level of an analyte in blood from which a blood specimenwas obtained.

FIG. 3 is a flowchart representing steps in a method for providing testresult information to a user.

FIG. 4 is a representation of a database record correlating test resultinformation with a test number.

FIG. 5 is a flowchart representing steps in a method for preparing adatabase of test results and test numbers.

FIG. 6 is a flowchart representing steps in a method for preparing adatabase of blood analyte levels, solution analyte levels, and solutionnormalizing analyte levels.

FIG. 7 is a representation of a database record for a databasecontaining blood analyte level information, solution analyte levelinformation and solution normalizing analyte level information.

FIG. 8 is a schematic illustration showing various communicationsbetween a customer, a results providing facility, and others inconnection with a testing protocol.

FIG. 9 is a perspective view of the obverse side of a blood collectiondevice useful in conjunction with the invention.

FIG. 10 is a perspective view of the reverse side of the device shown inFIG. 9.

FIG. 11 is a representation of a kit useful in conjunction with theinvention.

FIG. 12 is a perspective view of the obverse side of alternativeembodiment of a two-gang blood collection device in accordance with anembodiment of the invention, shown prior to use with one gang covered.

FIG. 13 is a perspective view of the obverse side of the bloodcollection device shown in FIG. 12, shown with the second gang exposed.

FIG. 14 is a side perspective view of the blood collection device shownin FIGS. 12 and 13, partially opened to show internal details.

FIG. 15 is an exploded view of a lancet useful in conjunction withpreferred embodiments of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is applicable to the testing of any specimen that ismodified from its original form prior to testing. Most commonly, thespecimen is a dried specimen, which has been dried to facilitate storageor transport of the specimen or for other purposes. In preferredembodiments of the invention, the specimen is a medical specimen, and inhighly preferred embodiments of the invention, the specimen is a bloodfluid specimen, by which is contemplated a dried blood spot, a driedserum spot (for instance, as obtained from the device disclosed in U.S.Pat. No. 5,435,970 or that shown in U.S. Pat. No. 4,839,296 issued toKennedy, et al. and assigned to Chem-Elec, Inc. of North Webster, Ind.),or another blood fluid specimen. The invention is applicable to thetesting of the modified specimen for any suitable purpose, and inparticular to testing for any analyte in the specimen. For instance,when the specimen is a blood fluid specimen, the test may be a test forprostate specific antigen (PSA), alanineamino transferase (ALT), lipids,such as triglycerides, high density lipoprotein (HDL), low densitylipoprotein (LDL), or any other analyte of interest. The invention isapplicable to the determination of the level of analyte in the originalspecimen, for instance, the level of total cholesterol in the blood fromwhich a blood fluid specimen has been obtained. The “level” of theanalyte can be expressed in any suitable units, such as molarconcentration, weight concentration, or the like. Blood serum isparticularly preferred, but it is contemplated that other fractions suchas cells, platelets, gamma globulins, plasma or the like may beemployed. For instance, it may be designed to test blood cells inconnection with a fasting plasma glucose test. More generally, any bodyfluid is susceptible to analysis in conjunction with the invention. Inlight of the foregoing, the preferred embodiments of the invention willbe further described with respect to the determination of the lipidprofile in a blood sample, but it should be understood that theinvention is not limited thereto.

The facility or other entity that performs the test of the blood fluidspecimen may or may not be the same entity that calculates the level ofthe analyte in the blood fluid specimen or the entity that receives aninquiry from a user and reports the test results to the user. To testthe blood fluid specimen, the specimen is first received by the testingentity and is eluted with a liquid, preferably deionized water. It iscontemplated that the liquid may be a non-aqueous liquid or may be anaqueous solution, preferably a solution that is free or essentially freeof sodium ions or any other normalizing analyte. Alternatively, thesolution may have a known amount of the normalizing analyte that can betaken into account during normalization. Preferably, when the testingentity is a testing facility that is intended to test numerousspecimens, the eluant is added in a standard amount, which typically is600 μl (0.6 ml). The eluant in some embodiments may be a bufferedelectrolyte solution.

After eluting the specimen, preferably the specimen first is tested forthe content of a normalizing analyte, such as sodium and chloridecontent, and in some embodiments osmolality, which generally representstotal content of sodium, glucose, and blood urea nitrogen (BUN). To testfor sodium and chloride, an ion specific electrode (ISE), such as thatsold by Orion may be employed. Preferably, information concerning boththe sodium and the chloride content of the solution are obtained, theinformation being, for instance, analog information such as anelectrical signal or digital information such as a printout representingthe sodium or chloride content or a digital signal containinginformation concerning the sodium or chloride content. Most preferably,osmolality also is measured. It should be noted that the invention isnot limited to the use of sodium or chloride as normalizing analytes,but to the contrary, any other analyte (which includes a property suchas osmolality) may be measured. It is contemplated in preferredembodiments that the sodium, chloride, and osmolality levels aremeasured against a predetermined range to determine whether the amountof serum is sufficient to perform an adequate test. For instance, it iscontemplated that for a cholesterol test, there ideally should be atleast approximately 15-17 μl of serum available for testing. If thesodium content of the eluted solution demonstrates that the serum levelis far outside this range, the specimen may be rejected as unsuitablefor testing. Generally, the specimen may be rejected if there isinsufficient serum in the solution, although it is contemplated that insome cases excess serum may be grounds for rejection. Persons skilled inthe art may determine how far outside of the desired range the contentof normalizing analyte may be allowed to vary without triggeringrejection of the specimen.

Before or after the levels of the normalizing analytes are determined(but preferably after), the solution can be split into four aliquots, or“channels.” Each channel is then respectively tested for triglyceridelevel, HDL level, LDL level, and in a preferred embodiment, ALT level(which may be of interest in informing a physician whether the patienthas an abnormal liver which would contraindicate the use of certaindrugs). The analyte levels are measured using any technique known in theart or otherwise found to be suitable. For instance, a cholesterol testis disclosed in Allain, C. C., Poon, L. S., Chan, G. S. G., Richmond,W., and Fu, P. C., Clin. Chem. 20:474-75 (1974); see also Roeschlau, P.Brent, E. and Gruber, W A., Clin. Chem. Clin. Biochem. 12:226 (1974). Atest for HDL is disclosed in RiFai, N., Warnick, G. R., Ed., LaboratoryMeasurement of Lipids, Lipoproteins, and Apolioproteins (1994). A testfor triglycerides is disclosed in McGowan, M. W., Artiss, J. D.,Strandbergh, D. R., Zak, B. Clin. Chem. 29:583 (1983). A test for theliver enzyme ALT is disclosed in Wroblewski, F., LaDue, J. S., Proc.Sec. Exp. Biol. Med. 34:381 (1956). The invention is not limited to theforegoing tests or analytes, but to the contrary is applicable to othertests for these or other analytes.

After the analyte levels have been measured, the level of at least oneanalyte (and preferably all analytes) in the blood from which the bloodfluid specimen was obtained is calculated or otherwise determined basedon the solution level of the analyte and on the solution level of atleast one normalizing analyte. It is contemplated that the calculationof a blood analyte level may be as simple as multiplying the solutionanalyte level by the ratio of the blood normalizing analyte level to thesolution normalizing analyte level, the blood normalizing analyte levelbeing estimated based on the mean of a normal population distribution.For instance, it is believed that the normal blood sodium level inhumans ranges from 136 to 142 mEq/L with a mean of 139 mEq/L and thenormal chloride level ranges from 95 to 103 mEq/L with a mean of 99mEq/L. It is contemplated that through the use of two normalizinganalytes, the blood analyte level may be determined by calculating theblood analyte level based on the first normalizing analyte level,calculating the blood analyte level based on the second normalizinganalyte level, and then calculating the mean average of the bloodanalyte levels thus determined.

If additional normalizing analytes are evaluated, the mean average ofall blood level analytes thus determined may be calculated; if desired,where there are at least two normalizing analytes, the average may beweighted towards a specific normalizing analyte. For instance, it iscontemplated that Bayesian statistical methods may be used to assign arelative weight to the blood analyte levels determined with reference toeach analyte. Such statistical techniques may take into account not onlythe absolute magnitude of the level of the normalizing analyte level butalso the difference between the actual level and the magnitude expectedbased on the expected amount of serum, and the standard deviation of thenormal population distribution of the analyte. These techniques,sometimes referred to as “maximum likelihood” or “prior probabilityanalysis” techniques, may be used to provide an approximation of theblood analyte level. Further testing concerning such statisticaltechniques may be found in Casella, G., Berger, R. L., StatisticalInference (1990) and Carlin, B. P., Louis, T. A., Bayes and EmpiricalBayes Methods for Data Analysis (2d Ed. 2000).

Further details concerning the distribution of sodium, chloride, andosmolality in the normal human population may be found in Ravel,Clinical Laboratory Medicine (6th Ed. 1995); see also Penney, M. D. andWalters, G., Ann. Clin. Biochem. 24:566-71 (1987) and Fraser, C. G.,Cummings, S. T. Wilkinsen, S. O. et al., Clin Chem. 35:783-86 (1985). Itis further contemplated that a more complicated function of solutionanalyte level and the levels of one or more normalizing analytes may beemployed to calculate the blood analyte levels.

With reference now to FIG. 1, the generalized method shown therein isapplicable where the same entity performs the test and calculates theblood analyte level. Thus in steps 101 and 102 respectively the ISE(e.g., sodium) is immersed into the solution, and sodium levelinformation is obtained. The steps are repeated for the receipt ofchloride information, as shown in steps 103 and 104. Informationconcerning the analyte of interest is received in step 105, and theblood analyte level is calculated in step 106. If, in step 107, it isdesired to test an additional analyte for the same specimen, controlpasses to step 105 where the solution analyte information is receivedfor the new analyte. It is contemplated that the steps of testing forthe analytes of interest and the normalizing analytes may be performedby one entity and that the calculation of the blood analyte level may beperformed by a separate entity. Thus, for instance, in FIG. 1, steps 101and 103 may be omitted if the entity calculating the blood analyte levelis not the same entity as the entity that performs the test. The methodoutlined in FIG. 1 is very general, and other steps may be added, stepsmay be omitted or performed in a different order, and more generally themethod may be otherwise performed. For instance, steps of elution andverifying proper serum level are not shown, but are preferably employed.

In calculating the analyte level, it has been discovered that it isoften desirable to add or subtract a “recovery delta” from the amount ofanalyte measured in solution, and to base the calculation of one analyterelative to normalizing analyte or analytes on the recovery deltacorrected solution analyte level. While it is not wished to be bound byany particular theory of operation, it is believed that, in someinstances, analyte will be lost on the strip. As a general matter, thetotal loss of analyte will be proportional to the amount of analyteoriginally present on the strip. In some instances, however, as theamount of analyte increases, the proportional analyte loss is believedto decrease. The analyte is thought to “saturate” potential bindingsites on the strip. The addition or subtraction of the recovery delta isintended to correct for this phenomenon.

In other cases, particularly in the measurement of HDL, certain analytescan give a “false positive” for which correction is desired. In the WakoHDL assay, HDL is detected after the LDL (and VLDL) molecules areprotected from enzymes by certain antibodies. The remaininglipoproteins, which are attacked by enzymes and analyzed, normally aredeemed to consist of solely HDL. This assumption generally holds truefor venous blood samples. However, elution from a blood collectiondevice in accordance with the invention sometimes yields partiallydenatured LDL and VLDL molecules. These molecules cannot be protected byantibodies and accordingly are susceptible to enzymatic attack in thesame fashion as is HDL, thus providing a false measurement of HDL. Therecovery delta thus is of the opposite sign than the recovery deltaheretofore described.

In light of the foregoing, it should be appreciated that the recoverydelta may be a positive value or a negative value. In accordance withsome embodiments of the invention, the inventive method is deemed toencompass the determination of a recovery delta for a particular analyteand set of testing conditions. The recovery delta may be estimated ormay be determined empirically, with reference to the data taken fromknown samples.

Another potential corrective factor takes into consideration the decayof the analyte, which is generally based on time since the sample hasbeen taken from a patient, and which sometimes depends on climactic orother environmental factors. In subtracting or adding a recovery deltato the measured solution analyte level, the recovery delta may bedetermined according to the following formula:A+B (time)wherein A is a positive, zero, or negative value and wherein B is apositive or negative value. Values A and/or B may be determined orestimated based on climactic conditions, to account for different decayrates. Alternatively, the recovery delta may be calculated as a functionof time and/or climactic conditions such as humidity, temperature, orthe like in a multivariate manner that may be first order or not firstorder with respect to any of such variables.

The preferred normalizing analytes are sodium and chloride. It has beendiscovered that the calculation of blood analyte levels based on sodiumand on chloride each may be made by assuming a linear relationshipbetween normalizing analyte levels and blood analyte levels. In otherwords, for sodium, the blood analyte level may be determined inaccordance with the following generalized equation:BAL=(solution analyte level−recovery delta)(A+BX)wherein BAL is calculated blood analyte level, X is the normalizinganalyte level, and A and B are calculated or empirically determined orotherwise predetermined constants. Surprisingly, however, it has furtherbeen found that the resulting analyte calibration curve often is notlinear. For instance, with respect to chloride, the calibration of theion specific electrode frequently underestimates chloride levels atconcentrations lower than 138 mEq/L, and frequently overestimateschloride levels higher than 138 mEq/L. Accordingly, in accordance withhighly preferred embodiments of the invention, calculation of analytelevel in the blood from which the blood fluid specimen was takenincludes a predetermined nonlinear chloride correction factor. In theembodiment of the invention, a nonlinear connection function may be usedfor chloride but not for sodium.

It is contemplated that the analyte level, first normalizing analytelevel, and second normalizing analyte level may be independentlydetermined and these values used to calculate the blood level of theanalyte. For instance, the cholesterol tests hereinbefore discussedtypically are performed via enzymatic techniques in which the opticaldensity of a solution is measured. The “cholesterol value” of thesolution then may be expressed as:CV _(s) =f(OD)wherein CVs, the solution cholesterol concentration, is calculated as afunction of the optical density, OD, when analytical reagents are addedto the sample in accordance with testing techniques known or otherwisefound to be suitable. The solution sodium concentration, or Na_(s), maybe used to calculate the blood cholesterol level, CV_(b), in thefollowing manner:CV _(b) =f(CV _(s), Na_(s))Numerous other forms of such calculations are possible. For instance, acorrection factor (CF) may be determined as a function of the solution'ssodium level, wherein:CV _(b) =f(CV _(s) , CF)andCF=f(Na_(s))

It is alternatively contemplated that a single apparatus or system maybe designed for the calculation of blood analyte levels, wherein ananalog or digital electrical signal is generated corresponding to thelevels of analyte and normalizing analyte in the solution. For instance,the blood cholesterol number may be calculated as a function of themagnitude of two electrical signals:CV _(b) =f(E ₁ , E ₂)wherein E₁ represents the magnitude of an electrical signal receivedfrom a spectrophotometer in measuring optical density for purposes ofevaluating total solution cholesterol level and E₂ represents themagnitude of an electrical signal received from an electrode specific tosodium.

In actual practice, it is contemplated that numerous variables willaffect the results obtained for a given set of specimens. For instance,the readings obtained from an ISE may “wander” from day to day, and thedevice used to collect the blood or other fluid specimen may containimpurities (such as sodium) that have the potential to introduce errorsinto the test. For this reason, from time to time a “tare” procedure maybe employed. Periodically, a plurality of specimens having a known ormeasurable analyte level is provided, and from these specimens areprepared modified specimens, the modified specimens being specimens asmodified in the manner expected of the unknown specimens. For instance,some number (e.g., six) blood specimens may be periodically placed ontoa blood spot collection device similar to those used in the field anddried, followed by elution of the dried samples to form solutions. Thesolutions are then tested for the level of the analyte and one or morenormalizing analytes. From these tests, an algorithm for determining theoriginal fluid analyte level as a function of the measured analyte leveland the levels of the normalizing analyte or analytes may be derived.Using this algorithm, modified fluid specimens may be analyzed, whereinthe levels of analyte and normalizing analyte may be measured, and thelevel of analyte in the original specimen may be determined as afunction thereof. Errors introduced by impurities (such as sodium) inthe collection device will be resolved by this methodology, and errorsintroduced by factors such as machine calibration will be resolvablewith periodic re-calculation of the algorithm. The tare procedure may beperformed occasionally or regularly at predetermined intervals (e.g.,every day, week, month, or year).

In accordance with the foregoing teaching, the following generalizedprotocol may be used in determining analyte levels. This protocolassumes that the analyte of interest is a cholesterol and that thenormalizing analytes are sodium and chloride.

Calibration of Equipment

Fifteen calibrators and four controls are evaluated. Each “calibrator”is a blood sample of known cholesterol concentration and sodium andchloride concentrations. For each of the calibrators, scalar values Xand Y are calculated in accordance with the following equations:Y=(Analyte−Analyte RD)/Expected AnalyteX={E×10 exponent [Log 10(E/DC)×((E Slope−FS)/FS]−EBG/EV}/Expected E

In the foregoing equations, the value “analyte” is the measured analyteconcentration, and the value “analyte RD” is the recovery delta. Theexpected analyte is the known analyte concentration. Value “E” is themeasured normalizing analyte (electrolyte) concentration; value “DC” isa drift control value which represents the center of the s-shapedcalibration curve and which, for chloride, is 138 mEq/L and for sodiumis 5 mEq/L. Value “E Slope” is a calibration slope determined based onthe assumption that the normalizing analyte calibration curve is linear.Value “FS,” or “Fixed Slope,” is a calculated correction factor, whichis calculated in accordance with the following equation:Ideal CL Slope=Cl Slope*[(Log 10(CL)−Log 10(138)]/[Log 10(ExpectedCL)−Log 10(138))]“Expected E” is the known normalizing analyte concentration Value “EBG”is electrolyte background, and value “EV” is elution volume.

After determination of x and y for each calibrator, scalar values A andB are determined:

$A = \frac{{( {{sum}\mspace{14mu} y} )( {{sum}\mspace{14mu} x^{2}} )} - {( {{sum}\mspace{14mu} x} )( {{sum}\mspace{14mu} x\; y} )}}{{N( {{sum}\mspace{14mu} x^{2}} )} - ( {{sum}\mspace{14mu} x} )^{2}}$$B = \frac{{N( {{sum}\mspace{14mu} x\; y} )} - {( {{sum}\mspace{14mu} x} )( {{sum}\mspace{14mu} y} )}}{{N( {{sum}\mspace{14mu} x^{2}} )} - ( {{sum}\mspace{14mu} x} )^{2}}$

These variables constitute the results of a regression analysis thatprovide, A (abscissa intercept) and B (a slope) for the calculation ofunknown samples and controls.

Once a calibration has been made, a sodium normalized blood analytelevel and a chloride normalized blood analyte level are calculated inaccordance with the following equations:

${{Na}\text{-}{Normalized}\mspace{14mu}{Analyte}} = \frac{( {{Analyte} - {{Analyte}\mspace{14mu}{RD}}} )}{\begin{matrix}( {{A1} + {{B1} \times \{ {{Na} \times 10\mspace{14mu}{exponent}} }}  \\\lbrack {{Log}\mspace{11mu} 10\mspace{11mu}( {{{Na}/{Na}}\mspace{14mu}{DC}} ) \times}  \\{ { {{( {{{Na}\mspace{14mu}{Slope}} - {{Na}\mspace{14mu}{FS}}} )/{Na}}\mspace{14mu}{FS}} \rbrack - {{Na}\mspace{14mu}{{BG}/{EV}}}} \}/} \\ {{Population}\mspace{14mu}{Mean}\mspace{14mu}{Na}} )\end{matrix}}$${{Cl}\text{-}{Normalized}\mspace{14mu}{Analyte}} = \frac{( {{Analyte} - {{Analyte}\mspace{14mu}{RD}}} )}{\begin{matrix}( {{A2} + {{B2} \times \{ {{Cl} \times 10\mspace{14mu}{exponent}} }}  \\\lbrack {{Log}\mspace{11mu} 10\mspace{11mu}( {{{Cl}/{Cl}}\mspace{14mu}{DC}} ) \times}  \\{ { {{( {{{Cl}\mspace{14mu}{Slope}} - {{Cl}\mspace{14mu}{FS}}} )/{Cl}}\mspace{14mu}{FS}} \rbrack - {{Cl}\mspace{14mu}{{BG}/{EV}}}} \}/} \\ {{Population}\mspace{14mu}{Mean}\mspace{14mu}{Cl}} )\end{matrix}}$

In these equations, the expected electrolyte value has been replaced bythe population electrolyte mean value, which for sodium is 146 mmol/Land for chloride is 107 mmol/L (as measured using this equipment).

Finally, the mean analyte level is determined by calculating the meanaverage of the sodium normalized blood analyte level and the chloridenormalized blood analyte level. It should be appreciated thatinnumerable variants of the foregoing procedure fall within the purviewof the invention.

It is noted that the correction factor for both sodium and for chlorideis logarithmically determined, although other non-linear correctionfactors may be employed. In either case, the calculation may proceed inaccordance with the following equations:

${{Na}\text{-}{Normalized}\mspace{14mu}{Analyte}} = \frac{( {{Analyte} - {{Analyte}\mspace{14mu}{RD}}} )}{( {{A1} + {{B1} \times {X1}}} )}$${{Cl}\text{-}{Normalized}\mspace{14mu}{Analyte}} = \frac{( {{Analyte} - {{Analyte}\mspace{14mu}{RD}}} )}{( {{A2} + {{B2} \times {X2}}} )}$

wherein A and B are scalar values that are calculated as heretoforedescribed or are calculated without accounting for non-linearcalibration factors and wherein X1 and X2 are respectively correctionvalues for sodium and for chloride. Values X1 and X2 may be calculatedas a linear function of normalizing analyte concentration, as alogarithmic function thereof, or as any other appropriate function.

The foregoing exemplary equations and procedures are not meant to beexhaustive but, to the contrary, are intended to illustrate thatinnumerable variants of the methods for calculating the blood analytelevel are included within the scope of the invention. For instance, withrespect to FIG. 2, in one such variant, an ISE (sodium) is immersed intoan eluted sample at step 201, and a signal corresponding to the sodiumlevel is received at step 202. The signal may be a digital signal, ormay be an analog signal, the level of which is recorded. At steps 203and 204, the same steps are repeated for chloride level, and at steps205 and 206 respectively, a test for the analyte is performed and asignal is received corresponding to the analyte level. At step 207, thesolution sodium level is calculated; at step 208, the chloride level iscalculated, and at step 209, the solution analyte level is calculated.At step 210, the blood analyte level is calculated, in this instancebased on the magnitude of the solution sodium level, the solutionchloride level, and the solution analyte level. If, at step 211, it isdesired to test for an additional analyte for the same specimen, controlpasses to step 205. In such case, if the solution sodium and chloridelevel have been stored, steps 207 and 208 may be omitted after a signalis received corresponding to the second analyte level. The process maybe controlled by any suitable microprocessor or microcontroller (notshown).

As stated hereinabove, it is contemplated that the entity who providestest results to a user, who may or may not be the health careprofessional who has ordered the test, in turn may be the same ordifferent entity from the entity which performs the calculation of theblood analyte level, which in turn may be the same or different entityfrom the entity which tests the specimen and generates informationcorresponding to the analyte level or levels and the normalizing analytelevel or levels. A very general protocol for a results providingfacility is set forth in FIG. 3, wherein an inquiry is received from auser at step 301, and the user is prompted for his or her test number atstep 302. At step 303, the test number is received, and at step 304, atest result database is queried for test result information. Theinformation is received at step 305 and is provided to the user at step306.

With further reference to FIG. 4, the test result database describedabove may be structured in any suitable manner. With respect to, forinstance, database record 400, the test result information 401, which inthe illustrated embodiment includes two items of information, bloodanalyte information 1 and blood analyte information 2, is correlatedwith the test number 402. The test number may be an anonymous testnumber or may be a test number that is associated with a user, forinstance, elsewhere in the database record 400 (not shown) or in adifferent database.

With reference to FIG. 5, the database may be prepared by creating adatabase record (shown in step 501), receiving test result informationand a test number (shown in steps 502 and 503 respectively) and, asshown in step 504, entering the test number and test result informationinto the database record. More information concerning the role of aresults providing facility in a medical or wellness testing protocol canbe found in the aforementioned Quattrocchi patents and in copendingapplication Ser. No. 09/709,884.

The invention additionally contemplates a method for preparing adatabase for use in calculating blood analyte levels. The blood analytelevel may be calculated with specific reference to the database, oralternatively the database may be used in conjunction with thepreparation of an algorithm for enabling blood level calculation. Thedatabase preferably is prepared with reference to blood having a knownlevel of cholesterol or other analyte of interest. Plural specimens ofblood having different levels of the analyte are then reduced to anmodified specimen, such as a blood spot or serum specimen, and eachspecimen is analyzed for the analyte of interest and for a normalizinganalyte. For instance, with respect to FIG. 6, a database record iscreated at step 601, and known blood analyte level information isreceived at step 602. Information as to the solution analyte level andthe level of two normalizing analytes, sodium and chloride, for example,are received at steps 603-605, and at step 606, the information receivedis entered into the database record. If, at step 607, an additionaldatabase record is to be created, control passes to step 601, wherein anew database record is created for the new specimen. It should be notedthat the order of the steps is not critical, and indeed the database maybe prepared sequentially with respect to each blood specimen (i.e., eachspecimen is reduced to an modified specimen, tested, and the resultsentered into a database record prior to altering the next specimen ofblood), sequentially with respect to database record (wherein all of theblood specimens are reduced to modified specimens prior to entering thefirst database records) or by any other suitable methodology. A databaserecord 700 as shown in FIG. 7 is thus prepared, with entries 701 through704 representing respectively blood analyte level, solution analyte,solution sodium level, and solution chloride level.

As discussed above, rather than being calculated, the blood analytelevel in a blood fluid specimen may be determined with reference to thedatabase, for instance, by finding the solution analyte level andsolution normalizing analyte level or levels in the database that areclosest to those of the specimen. Alternatively, any suitablestatistical or mathematical technique may be used to derive an algorithmfor calculating the blood analyte level from the solution analyte leveland at least one normalizing analyte level. In some embodiments, thealgorithm is first order with respect at least to the solution analytelevel, and may be first order with respect to the solution analyte leveland one or both normalizing analyte levels.

The invention preferably is conducted in accordance with the generalschematic set forth in FIG. 8. Generally the customer 801 purchases atest kit from a physician or retail store 802 (transfer of the kit isshown via transfer communication 805) or in other embodiments a patientis provided with a test kit by or at the direction of a health careprovider. The test kit (not shown in FIG. 8) preferably includesinstrumentalities for allowing the customer to obtain a blood, serum orserum spot specimen. For instance, as discussed more fully in theaforementioned Quattrocchi patents, the test kit may include a lancetfor pricking the user's finger, a blood spot card, or serum spot card,(or the device shown in subsequent figures hereinafter discussed) aninformed consent form, and a test number. After preparing the blood,serum or serum spot card, the customer sends the dried blood specimen toa results providing facility 803 as shown via transfer communication806. In the illustrated embodiment, the results providing facility 803sends the specimen to a separate testing facility 804, as shown viatransfer communication 809. As shown via communication 810, the testingfacility provides the test results to the results providing facility.The results may be “raw” results, i.e., results in which the level ofthe analyte in the blood has not been determined or obtained, oralternatively the testing facility may calculate the blood analyte leveland report that result to the results providing facility. As shown atcommunication 807, the customer contacts the results providing facility,and at communication 808, the results providing facility provides thetest results to the customer. Optionally, the results providing facilitymay be equipped to communicate directly with the physician's office, asshown at communications 811 and 812. Except where transfer of a physicalspecimen is required, the communication may be made via any means ormethod now known or hereinafter discovered, for instance, via telephone,wireless communication, electronic mail or “chat” or other electroniccommunication, or other form of communication.

With reference now to FIGS. 9 and 10, the illustrated fluid collectiondevice 900 includes two gangs 901, 901, each comprising a fluidcollector 903, 904 that is disposed between a superstrate sheet 905 anda substrate sheet 906 and that is generally fixed with respect to thesuperstrate sheet 905. The fluid collector is ordinarily connected tothe substrate sheet 906 (a portion of which is visible) at one end 907,908, although the collector may be flexible and thus not entirely fixedwith respect to the substrate sheet 905. The substrate is provided withat least one aperture (two shown as 909, 910) by which a user mayfluidically transfer blood to the collector. In the illustratedembodiments, secondary apertures 911, 912 are provided. To use thedevice, a user dispenses blood onto the collector, whereby some or allof the blood wicks in the direction shown by arrow 913 until theportions 914, 915 of the collectors 903, 904 visible through thesecondary apertures 914, 915 become tinted, whereupon the user isprovided with an indication that sufficient blood has been collected. Asillustrated, the device preferably is disposed horizontally relative tothe ground during the wicking of blood. Any other suitable indicatorthat an amount of blood predetermined to be adequate may be provided. Inthe illustrated embodiments, instructions 917 are provided on thesubstrate sheet 905 and identification information spaces 918 (shown inFIG. 10) are provided on the substrate sheet 906. The device may beprovided with non-textual machine-readable indicia (such as barcode 919)or textual indicia that indicates, for instance, a test number, codenumber, lot number, or other desired information.

With reference to FIG. 11, the illustrated kit 1100 includes thespecimen collection device 900 illustrated in FIG. 9, and numerous othercomponents, some or none or all of which in practice may be included ina kit. The kit includes a barrier pouch 1103, a desiccant pouch 1104, alancet 1102, and instruction sheet separate from the kit, a results formfrom a previous test, and a requisition form as specifically shown inthe figure. In preferred embodiments, the kit includes a mailing device,most preferably a preaddressed envelope with postage prepaid for sendingthe collection device to a testing facility or other appropriatefacility. In practice, the kit may further include a bandage, gauze pad,and alcohol pad for use with drawing blood from the patient (not shown)and a form for providing informal consent, which informed consent may beprovided anonymously as described in the heretofore mentionedQuattrocchi patents. The barrier pouch should be a pouch that iseffective in protecting the dried blood sample during shipping. Onesuitable barrier material is sold by Caltex Plastics of Vernon, Calif.and comprises a multi-layer barrier film consisting of 25 bleach MGPaper, 48 GA polyester film, 0.0005 aluminum foil, and 0.003 EVAco-polymer, the layers being adhesively bonded together. The pouchpreferably is formed with at least one self-sealing device, such as a“zipper” disposed at at least one end of the pouch. A pouch thatincludes two self-sealing devices, one at each end of the pouch,alternatively may be provided.

The desiccant pouch should be a porous container that includes suitabledesiccant effective to provide a desiccating protective effect on ablood fluid specimen, and to some extent to protect the integrity of thecollection device during transport to the physician or patient. Anysuitable desiccant material may be used in conjunction with theinvention. One suitable desiccant is made by SudChemie of Balen, N.Mex.under part number 4286. This material comprises silica and clay disposedin admixture in a 5 gram pouch. Any other suitable desiccant may be usedin conjunction with the invention.

Likewise, any suitable lancet may be employed in conjunction with theinvention. The illustrated lancet 1102 preferably comprises ablood-obtaining lancet such as that presently available from Palco Labsof Santa Cruz, Calif. as the EZ-LETS II or the larger lancet shown inFIG. 15. These devices are a single-use lancet that are spring-loaded toenable the lancet to sharply pierce a user's skin. Any other suitablelancet may be used in conjunction with the invention. The barrier filmpouch is sized to receive the fluid collection device. Preferably, thepouch is sized to receive the desiccant pouch and the fluid collectiondevice.

An alternative embodiment of a fluid collection device 1200 suitable foruse in conjunction with the invention includes two gangs 1201, 1202(gang 1202 not shown in FIG. 12). Each of the gangs comprises a fluidcollector that is disposed between a superstrate sheet 1205 and a devicesubstrate sheet 1206 (best shown in FIG. 14) and wherein each fluidcollector is generally fixed with respect to the superstrate sheet 1205.The fluid collectors are ordinarily connected to the substrate sheet atone end of the substrate sheet. In the embodiment illustrated in FIGS.12-14, usage instructions 1207 are provided on a portion of thecollector, the usage instructions including an illustration as to properand improper blood filling profiles.

The collector 1200 includes a tab cover 1208 that releasably covers oneof the gangs 1202. In ordinary use, the fluid collector is provided in aform such that the cover is closed. The user dispenses blood through afirst aperture 1210 in the first gang 1201 onto collector 1211, and,after the fluid collector becomes sufficiently filled, the user opensthe cover and repeats the dispensation of blood onto the second gang1202. Instructions to this effect are provided.

Each gang is provided with a second pair of apertures 1301, 1302, and,in accordance with this embodiment of the invention, each of the secondapertures is provided with a window 1303, 1304. The window allows for avisual indication as to fluid flow across the collectors but the windowinhibits blood flow through the second apertures 1301, 1302.

With particular reference to FIG. 14, the device is provided with aspacer 1401 disposed between the substrate and superstrate sheets 1205,1206 and which inhibits a contact of at least a portion of the substrateand superstrate sheets. In this embodiment of the invention, thesubstrate and superstrate sheets preferably are formed of afluid-resistant, laminated cardstock. In accordance with the heretoforedescribed construction, blood is inhibited from leaking off of the fluidcollectors onto the cardstock, and the presence of the spacer allows fora small air gap between the substrate and superstrate sheets to therebyassist in blood drying. As heretofore described with respect to otherembodiments of the invention, the device may be provided withnon-textural machine-readable indicia, such as a bar code, or textualindicia that indicates, for instance, a test number, code number, lotnumber, or other desired information.

An instruction set may be included as a separate sheet within the kit,or alternatively the instructions may be integral with (for example,imprinted on) the fluid collection device. The kit may further includeresults from a previous test. Such is useful, for example, in the caseof patients who require periodic testing, for instance, of bloodcholesterol. The invention encompasses in some embodiments a method ofproviding a test kit and test results to a health care provider and/or apatient, the test results being results from a previous test and thetest kit being a kit as heretofore described. In some embodiments, thepatient responds to an indication in the results form as to whether orwhen to obtain a subsequent blood sample or other type of sample.

The invention contemplates methods wherein a physician is provided witha test kit as heretofore described and wherein the patient's blood isdrawn at the direction of the physician or other health care provider,either at the premises of the health care provider or elsewhere withoutthe healthcare provider being present. In keeping with theseembodiments, the kit may include a requisition form, the requisitionform permitting indication of the type of test or tests to be conductedon the fluid to be collected by the device. In some embodiments, therequisition form lists a plurality of test types, and the healthcareprovider need only indicate (such as with a check mark) the type of testdesired. On any such form, space may be indicated for the health careprovider to indicate any other sort of test desired to be conducted.

In a highly preferred embodiment of the invention, the fluid collectoris an absorbent paper or glass fiber substrate that is coated with asaccharide, preferably a mono or di-saccharide and most preferablyxylose. The saccharide should be present in contact with the substratein an amount effective to inhibit triglycerides ordinarily present inthe expected blood sample from binding to the fiber matrix. Thesubstrate should be one that permits at least substantial separation ofthe red blood cell component of blood cells from other portions of theblood (i.e., serum). It is believed that the saccharide componentpermits more effective recovery of the serum components from thesubstrate sheet. The substrate may be coated only at the surface on oneor both sides with the saccharide, but preferably the substrate iscoated on internal surfaces as well as on the exterior surface. In oneembodiment, 180 μl of a 5% solution of xylose is applied to the internalsurface of the 0.8×7 cm substrate (such that substantially all of thesubstrate is wetted) and allowed to air dry. If the fluid collector isused in the device shown in FIGS. 9 and 10, the blood cells will remainnear the end of the collection device (opposite the direction of arrow913) while the serum will wick toward the other end of the card. Uponreceipt by a testing laboratory, a portion of the fluid collector may beexcised and eluted. Preferably, the excised portion includes a portionof the collector “above” the terminal wicking point of the serum. It hasbeen found that using a two-gang device as described herein, if one orboth of the fluid collectors are deemed to have been filledinadequately, one-half of each fluid collector may be excised andeluted. One commercial product (Whatman GF/AVA paper) contains sodium,and it is believed that by excising filter paper above the terminalwicking point a consistent amount of sodium will be introduced into theeluted fluid. The device may be prepared by applying a solution of thesaccharide to the substrate.

The glass fiber paper heretofore described comprises a mat of glassfibers that are at least substantially coated with polyvinyl alcohol.The fibers define a plurality of pores that have a pore size that, inpreferred embodiments of the invention, is effective to at leastsubstantially prevent lysing of red blood cells while permitting atleast substantial separation of serum from red blood cells viadifferential wicking. Any suitable substrate that provides such a poresize and that permits such substantial separation in the absence ofblood cell lysing may be used in conjunction with the invention.Preferably, the average pore size defines a fluid removal rating, asthis term is used in conjunction with filtration technology, of 1.7micron.

The invention enables venous blood analyte levels to be determined fromcapillary blood specimens. It is contemplated that in most embodimentsthe solution analyte level will be normalized to the venous blood levelof the analyte, but it is also contemplated that the solution value maybe normalized to capillary blood level (or for that matter a differentblood level).

The databases discussed herein may be created and stored as computerfiles on a computer readable medium, such as a diskette, hard disk,CD-ROM, DVD-ROM, ROM chip or EPROM chip, or any other suitable medium asmay be now known or hereinafter discovered. The tests for the analyteand normalizing analytes may be performed by any conventional orotherwise suitable technique now or hereinafter found to be suitable,and likewise the analyte and normalizing analyte (which may be discreteatoms, ions, compounds, biochemical materials, or properties) may bethose specifically described herein or others as may be found suitablefor use in conjunction with the invention.

The following examples are provided to illustrate the invention, butshould not be construed as limiting the invention in scope unlessotherwise indicated. Unless otherwise indicated in these examples, themeasured analyte level was corrected using sodium as the solenormalizing analyte. The correction was made using a simple linearregression. It should be understood that more complex single variableand multivariate regressions may be used in conjunction with theinvention, and thus the statistical techniques employed in theseexamples should be viewed as non-limiting.

EXAMPLE 1

This example demonstrates the performance of the invention in themeasurement of total cholesterol.

Fifteen patients were used to obtain blood specimens (micro-serumspecimens) via venal puncture. Serum from each specimen was spotted anddried on filter paper with applied volumes ranging from approximately 8to 16 μl. The number of spots for each blood specimen is listed in thecolumn “No.” in the table below. Each spot was eluted and measured forcholesterol and sodium. For each specimen for each patient, thenormalized cholesterol level was calculated based on the level of ameasured analyte in the fluid (cholesterol) and a normalizing analyte(sodium). The normalized cholesterol level was obtained according to thepresent invention using linear regression techniques to yield thefollowing function: Normalized Cholesterol=MeasuredCholesterol/((−0.003306)+0.9781×(Measured Sodium/13)), where 139 (mEq/L)is the population mean for sodium. The regression was calculated basedon five direct measurements of the cholesterol level from the same bloodsample, as listed in the column “Mean Serum Cholesterol.” The meanaverage of the normalized cholesterol values for each patient is givenin the column “mean normalized cholesterol” and the coefficient ofvariation of the normalized cholesterol levels obtained for each patientis listed in the column designated “Normalized Cholesterol CV %.”

Mean Normalize Mean Serum Normalized Cholesterol Patient No. CholesterolCholesterol CV % A 11 152.35 153.68 3.85 Ja 12 165.79 162.50 1.42 Il 14180.93 180.47 4.61 Ca 12 186.20 182.28 0.70 Br 10 187.06 185.35 2.93 Mi12 187.14 186.21 1.85 Gr 12 187.42 189.14 1.65 Ed 12 200.38 197.18 1.36Tr 11 220.83 221.89 2.00 Bb 11 232.65 233.06 1.89 Ma 11 236.73 245.531.02 Jo 11 237.37 237.24 1.95 JJ 14 262.41 259.24 1.75 Kt 12 264.30268.23 1.86 TT 13 269.36 273.53 2.79

A comparative linear regression was generated for the data pointscollected in this Example. The linear fit followed the followingequation:Mean Normalized Cholesterol=−7.97+1.04×Mean Serum Cholesterol,with the correlation coefficient, expressed as R², being greater than0.99.

EXAMPLE 2

This example demonstrates the performance of the invention in themeasurement of HDL.

The same dried spots from the same fifteen patients in Example 1 wereused to obtain a measured value for HDL. The normalized HDL level wasobtained according to the present invention using linear regressiontechniques yielding the following function:Normalized HDL=HDL/(0.0158+1.060×(Sodium/139)). The following data wasmeasured or calculated in the same manner as in Example 1.

Mean Serum Mean Normalized Normalized Patient No. HDL HDL HDL CV % II 1445.77 47.03 2.35 A 11 46.05 47.77 2.17 Jo 11 47.40 48.50 2.12 Ja 1248.87 53.22 2.23 JJ 14 49.07 48.15 1.68 Gr 12 49.64 52.45 1.62 Mi 1259.96 58.95 1.69 Br 10 57.20 55.83 2.66 Ed 12 71.00 71.09 0.92 Kt 1273.08 72.46 1.53 TT 13 76.16 75.77 2.27 Ca 12 78.01 75.93 1.50 Bb 1178.77 73.35 1.99 Ma 11 87.84 84.75 0.94 Tr 11 91.15 86.42 1.46

A comparative linear regression was generated for the data pointscollected in this Example. The linear fit followed the followingequation:Mean Normalized HDL=8.15+0.87×Mean Serum HDL,with the correlation coefficient, expressed as R², being greater than0.99.

EXAMPLE 3

This example demonstrates the performance of the invention in themeasurement of triglycerides (TG).

The same dried spots from the same fifteen patients in Example 1 wereused to obtain a measured value for TG. The normalized TG level wasobtained according to the present invention using linear regressiontechniques yielding the following function:Normalized TG=TG/((−0.0136)+0.9307×(Sodium/139)). The following data wasmeasured or calculated in the same manner as in Example 1.

Mean Normalized Normalized Patient No. Mean Serum TG TG TG CV % Ca 1237.63 38.76 1.95 Bb 11 46.86 48.55 1.75 A 11 48.75 50.16 2.73 Ja 1249.68 49.94 3.31 Kt 12 52.15 48.19 1.32 Br 10 55.00 56.56 4.14 Ma 1156.05 56.40 2.03 II 14 59.09 60.88 6.22 Ed 12 62.91 61.65 1.25 Tr 1166.69 67.66 1.63 TT 13 68.76 72.14 13.37 Mi 12 71.84 72.63 1.62 Jo 11109.28 107.10 2.27 JJ 14 117.31 112.24 5.03 Gr 12 139.47 136.74 2.13

A comparative linear regression was generated for the data pointscollected in this Example. The linear fit followed the followingequation:Mean Normalized TG=3.36+0.95×Mean Serum TG,

-   -   with the coefficient, expressed as R², being greater than 0.995.

EXAMPLE 4

This example demonstrates the performance of the invention in themeasurement of LDL. The same observations from the same fifteen patientsin Example 1, 2 and 3 were used to calculate a value for LDL in serumand a value for LDL in MSS according to the Friedewald formula:Mean Serum LDL=Mean Serum Cholesterol−Mean Serum HDL−Mean Serum TG/5Mean Normalized LDL=Mean Normalized Cholesterol−Mean Normalized HDL−MeanNormalizedTG/5, respectively.The following data was calculated (mean serum LDL was calculated fromthe mean values reported in Examples 1-3)

Mean Serum Mean Normalized Normalized Patient No. LDL LDL LDL CV % A 1196.55 95.30 5.36 Ca 12 100.66 98.82 1.17 Ja 12 106.98 99.22 2.32 Gr 12109.88 109.00 2.19 Mi 12 115.81 112.90 2.80 Tr 11 116.35 121.21 3.11 Ed12 116.80 113.76 1.98 Br 10 118.86 118.21 3.23 Il 14 123.34 119.75 5.72Ma 11 137.68 149.45 1.72 Bb 11 144.51 150.01 2.12 Jo 11 168.11 167.552.66 Tt 13 179.45 183.33 3.33 Kt 12 180.78 186.13 2.19 JJ 14 189.88189.54 1.89

A comparative linear regression was generated for the data pointscollected in this Example. The linear fit followed the followingequation:Mean Normalized LDL=−8.16+1.07×Mean Serum LDL,

-   -   with the correlation, expressed as R², being equal to 0.98.

EXAMPLE 5

This example demonstrates the performance of the invention in themeasurement of total cholesterol.

One hundred thirty-two patients were used to obtain blood via venalpuncture (venous blood specimens) and by pricking their fingers(capillary blood specimens). Capillary blood was spotted onxylose-coated Whatman GF/AVA filter paper, using a device similar tothat shown in FIG. 9. Capillary blood specimens were dried and theportion of the filter paper which contained separated serum was cut outand eluted. Eluate from each specimen was measured for cholesterol andsodium. The normalized cholesterol level was obtained according to thepresent invention using a variable formula: NormalizedCholesterol=Measured Cholesterol/(A+B×(Measured Sodium/139)). In thisequation, A and B were scalar values that were periodically recalculatedbased on the “tare”procedure heretofore described, whereby a regressionfor six patients was calculated and the A and B values from thisregression were used to calculate normalized cholesterol values forspecimens analyzed before the next tare period. Actual (directlymeasured in venous blood) and calculated normalized cholesterol valvesfor these patients are given below.

Patient Serum Cholesterol Normalized Cholesterol 1 172.68 157.54 2149.25 154.61 3 176.81 175.60 4 189.78 187.41 5 170.38 173.03 6 189.67188.80 7 130.52 128.80 8 266.76 276.31 9 151.29 152.49 10 219.86 211.2311 242.00 251.07 12 232.41 230.66 13 173.09 176.48 14 190.89 190.86 15264.47 260.46 16 236.18 244.49 17 272.58 279.76 18 240.29 228.83 19169.32 166.57 20 192.02 195.03 21 239.83 235.33 22 225.13 225.13 23169.40 156.05 24 197.93 183.67 25 151.59 146.26 26 235.43 247.88 27178.84 170.79 28 196.40 191.34 29 240.99 230.52 30 171.53 173.95 31229.43 229.43 32 217.54 223.84 33 187.23 183.58 34 175.68 173.95 35174.69 172.34 36 251.23 249.20 37 203.70 185.98 38 123.30 114.96 39136.04 127.97 40 251.33 243.27 41 216.14 218.02 42 145.14 156.86 43208.58 203.43 44 250.25 245.07 45 235.76 250.40 46 193.19 187.83 47211.75 223.38 48 221.15 226.04 49 199.41 196.35 50 249.35 259.44 51166.46 165.63 52 154.64 151.56 53 187.36 190.37 54 256.78 260.40 55230.59 222.39 56 208.57 224.14 57 183.92 181.28 58 159.73 156.20 59155.31 153.59 60 205.29 197.61 61 204.49 198.97 62 219.21 221.45 63122.83 114.88 64 175.13 176.48 65 201.35 211.70 66 216.66 209.09 67227.50 231.96 68 151.28 153.23 69 130.10 128.40 70 175.95 173.45 71182.38 183.21 72 201.03 195.89 73 175.86 189.73 74 146.10 149.88 75116.17 103.88 76 193.58 197.59 77 291.91 296.11 78 184.93 185.49 79145.82 141.34 80 182.73 180.78 81 175.84 170.03 82 148.99 151.67 83212.79 213.40 84 228.82 225.39 85 218.44 229.26 86 169.43 173.84 87151.43 157.96 88 217.96 218.63 89 239.39 244.11 90 148.62 152.86 91136.81 132.60 92 119.13 113.31 93 121.10 119.61 94 165.31 163.34 95111.65 132.34 96 190.25 184.44 91 201.78 206.49 98 133.26 137.69 99225.84 221.61 100 244.66 230.25 101 164.72 168.10 102 150.75 146.82 103163.51 110.41 104 196.06 198.89 105 213.32 206.01 106 186.62 183.13 107163.46 162.71 108 244.58 250.24 109 231.82 231.32 110 171.94 172.27 111201.12 209.36 112 205.41 209.00 113 157.54 156.02 114 191.41 190.59 115192.20 197.31 116 193.52 183.12 117 257.83 248.49 118 178.32 171.44 119203.64 209.32 120 210.36 230.25 121 207.14 220.04 122 200.05 205.38 123216.34 219.09 124 190.10 179.14 125 293.34 272.48 126 228.57 226.02 127111.60 174.88 128 142.80 148.94 129 197.16 205.05 130 220.50 218.43 131220.32 231.50 132 255.18 255.23

A comparative linear regression was generated for the data pointscollected in this Example. The linear fit followed the followingequation:Normalized Cholesterol=−1.16+1.00×Serum Cholesterol, with thecorrelation coefficient, expressed as R², being 0.966.

EXAMPLE 6

This example demonstrates the performance of the invention in themeasurement of HDL. The dried spots and venous blood specimens from thesame one hundred thirty-two patients in Example 5 were used to measureHDL in capillary blood and compare it to a measured value for HDL invenous blood. The normalized HDL level in capillary blood was obtainedaccording to the present invention using a formula: NormalizedHDL=Measured HDL/(A+B×(Measured Sodium/139)), where A and B wereobtained as previously described. The following results were observed.

Patient Serum HDL Normalized HDL 1 58.90 61.12 2 41.28 42.33 3 38.5439.15 4 48.84 46.19 5 61.56 54.98 6 52.68 48.79 7 47.69 45.15 8 34.6939.49 9 57.45 56.32 10 38.00 36.33 11 47.53 42.14 12 60.04 58.94 1336.08 37.35 14 46.09 48.37 15 42.22 42.82 16 34.70 38.98 17 55.76 55.7918 21.16 24.53 19 55.33 55.69 20 44.66 42.65 21 83.26 81.00 22 44.3346.14 23 40.71 40.69 24 47.24 43.98 25 49.46 47.71 26 44.37 43.30 2750.16 48.34 28 55.49 61.30 29 58.90 61.12 30 41.28 42.33 31 38.54 39.1532 48.84 46.19 33 61.56 54.98 34 52.68 48.79 35 47.69 45.15 36 34.6939.49 37 57.45 56.32 38 38.00 36.33 39 47.53 42.14 40 60.04 58.94 4136.08 37.35 42 46.09 48.37 43 42.22 42.82 44 34.70 38.98 45 55.76 55.7946 21.16 24.53 47 55.33 55.69 48 44.66 42.65 49 83.26 81.00 50 44.3346.14 51 40.71 40.69 52 47.24 43.98 53 49.46 47.71 54 44.37 43.30 5550.16 48.34 56 55.49 61.30 57 49.27 45.94 58 51.73 51.78 59 38.07 36.9860 38.22 38.49 61 43.57 45.05 62 54.16 51.46 63 38.66 34.13 64 50.1448.65 65 57.94 54.11 66 46.02 44.67 67 49.21 52.36 68 43.15 45.31 6937.20 38.42 70 49.66 50.00 71 63.00 65.28 72 79.92 79.17 73 37.12 44.5774 59.14 60.35 75 32.49 28.57 76 56.08 59.37 77 64.22 70.04 78 46.5448.66 79 37.68 37.28 80 75.41 74.70 81 44.06 44.73 82 40.65 40.88 8393.40 91.97 84 40.97 47.04 85 69.63 75.17 86 36.13 38.81 87 34.88 36.4288 43.90 49.40 89 63.29 66.41 90 49.21 49.65 91 29.54 31.27 92 49.3049.87 93 35.82 34.39 94 49.66 51.20 95 39.01 39.79 96 36.92 34.49 9743.40 43.45 98 48.70 45.97 99 42.15 41.04 100 59.09 55.11 101 49.4647.04 102 33.36 29.81 103 49.36 47.93 104 43.02 39.12 105 39.81 41.06106 60.29 56.62 107 59.84 55.33 108 84.77 82.31 109 55.20 55.72 11054.77 56.06 111 69.16 67.30 112 38.18 40.50 113 37.11 36.49 114 51.3149.24 115 39.69 42.54 116 61.17 56.56 117 29.94 30.25 118 75.50 77.62119 56.94 57.49 120 68.89 71.30 121 37.89 40.82 122 73.57 72.14 12378.31 78.16 124 48.88 47.45 125 83.96 79.26 126 95.12 92.48 127 51.4452.50 128 38.88 38.10 129 41.70 44.58 130 47.80 46.24 131 56.42 59.35132 55.14 56.98

A comparative linear regression was generated for the data pointscollected in this Example. The linear fit followed the followingequation:Normalized HDL=2.47+0.953×Serum HDL,with the correlation coefficient, expressed as R², being greater than0.96.

EXAMPLE 7

This example demonstrates the performance of the invention in themeasurement of triglycerides (TG). The dried spots and venous bloodspecimens from the same one hundred thirty-two patients in Example 5were used to measure TG in capillary blood and compare it to a measuredvalue for TG in venous blood. The normalized TG evel in capillary bloodwas obtained according to the present invention using the formula:Normalized TG=Measured TG/(A+B×(Measured Sodium/139)), where A and Bwere obtained as previously described. The following results wereobserved.

Patient Serum TG Normalized TG 1 73.24 55.65 2 97.89 97.31 3 45.26 38.384 70.31 60.30 5 119.71 119.33 6 105.97 100.56 7 77.47 73.30 8 220.18236.94 9 191.79 203.18 10 177.10 177.03 11 112.19 116.71 12 73.24 55.6513 97.89 97.31 14 45.26 38.38 15 70.31 60.30 16 119.71 119.33 17 157.70164.69 18 122.09 124.56 19 66.86 63.24 20 138.31 151.08 21 146.08 137.3622 95.85 97.05 23 77.27 60.69 24 85.44 82.87 25 86.25 77.32 26 112.51110.68 27 176.25 184.16 28 190.63 189.57 29 95.17 98.92 30 98.52 98.7631 102.13 97.07 32 117.77 128.91 33 123.08 125.56 34 135.72 132.69 3576.46 71.14 36 230.90 210.77 37 80.41 67.66 38 99.43 85.63 39 86.8791.07 40 125.01 120.98 41 362.90 322.04 42 132.98 118.47 43 83.21 75.4344 52.45 53.34 45 53.91 50.52 46 349.76 357.87 47 135.25 139.57 48209.20 208.33 49 374.36 386.86 50 74.90 79.81 51 395.31 399.34 52 56.3854.87 53 217.08 258.78 54 52.83 71.35 55 136.53 144.81 56 115.98 118.4557 78.41 62.45 58 70.38 65.13 59 91.00 68.59 60 180.98 179.72 61 163.32188.88 62 72.16 65.05 63 102.89 101.45 64 50.24 49.05 65 184.45 195.4266 183.07 194.25 67 65.28 65.04 68 111.40 109.43 69 67.25 87.27 70 74.9272.25 71 100.19 105.33 72 136.82 132.52 73 119.29 129.90 74 119.76119.83 75 121.90 125.90 76 75.55 80.65 77 74.44 89.06 78 226.78 243.0579 71.19 78.23 80 98.89 93.66 81 127.93 135.56 82 333.65 352.31 83 97.1891.96 84 139.77 133.20 85 73.23 72.05 86 160.00 148.64 87 131.69 133.4988 69.07 66.79 89 271.22 248.43 90 91.86 98.00 91 231.14 224.76 92153.65 171.85 93 115.95 107.16 94 263.50 257.68 95 95.38 92.85 96 143.96125.21 97 110.10 131.36 98 97.72 93.75 99 158.22 151.23 100 123.80127.26 101 279.56 271.61 102 192.26 176.02 103 59.41 59.23 104 197.04186.32 105 182.29 170.98 106 96.16 91.53 107 80.46 72.56 108 65.55 68.16109 215.37 210.92 110 186.09 191.14 111 96.41 96.52 112 78.68 80.54 11383.96 73.13 114 207.32 208.03 115 37.41 37.32 116 103.17 93.38 117193.21 210.21 118 119.46 103.27 119 67.57 58.99 120 119.56 117.34 12175.42 52.90 122 311.18 315.01 123 67.72 68.28 124 127.36 129.28 12559.82 64.57 126 85.54 83.90 127 43.24 41.49 128 85.09 78.05 129 95.1599.45 130 92.21 75.05 131 72.46 88.51 132 56.52 57.13

A comparative linear regression was generated for the data pointscollected in this Example. The linear fit followed the followingequation:Normalized TG=−2.5+1.01×Serum TG,

-   -   with the coefficient, expressed as R², being 0.98.

EXAMPLE 8

This example demonstrates the performance of the invention in themeasurement of LDL. The same observations from the same one hundredthirty-two patients in Example 5, 6 and 7 were used to calculate a valuefor LDL in serum and a value for LDL in MSS according to the Friedewaldformula:Serum LDL=Serum Cholesterol−Serum HDL−Serum TG/5Normalized LDL=Normalized Cholesterol−Normalized HDL−Normalized TG/5.The following results were calculated:

Patient Serum LDL Normalized LDL 1 110.85 101.31 2 97.93 101.60 3 109.82103.89 4 110.51 112.89 5 108.21 107.74 6 126.07 121.49 7 49.76 54.13 8173.19 173.53 9 78.72 77.71 10 129.52 119.61 11 174.74 180.58 12 149.04140.09 13 98.72 101.13 14 115.22 115.25 15 187.28 180.56 16 146.29158.21 17 195.20 200.00 18 167.30 161.22 19 101.87 99.92 20 122.26127.45 21 168.27 164.79 22 149.27 148.28 23 100.92 85.06 24 129.40115.93 25 92.09 88.71 26 165.02 175.92 27 114.88 104.61 28 94.16 90.6229 154.94 142.87 30 114.95 117.39 31 144.71 148.13 32 152.62 164.12 33105.78 111.48 34 105.62 106.95 35 101.99 102.99 36 143.96 145.31 37119.65 105.96 38 68.65 63.55 39 78.02 75.16 40 180.50 174.23 41 110.10109.10 42 72.00 80.58 43 124.52 118.94 44 140.68 128.72 45 165.02 178.6646 92.96 83.43 47 145.15 156.72 48 133.07 131.63 49 105.59 101.08 50177.71 184.34 51 102.56 101.25 52 91.72 95.09 53 123.82 129.64 54 194.21203.38 55 144.23 138.11 56 120.42 125.06 57 120.22 122.32 58 87.42 84.1359 107.19 106.80 60 130.18 120.03 61 124.30 115.07 62 151.99 156.98 6361.89 58.86 64 111.54 110.38 65 128.43 143.14 66 150.60 143.35 67 150.93153.10 68 84.28 81.94 69 68.95 66.01 70 101.92 98.27 71 104.27 101.79 72106.22 98.91 73 93.38 96.56 74 72.72 73.88 75 63.90 56.58 76 111.92111.11 77 160.96 155.60 78 118.95 118.43 79 80.19 77.42 80 92.68 91.6781 99.78 95.56 82 82.00 84.09 83 105.58 108.07 84 133.61 128.66 85130.44 134.49 86 87.08 90.07 87 85.82 87.16 88 150.87 147.80 89 123.40126.22 90 80.33 84.64 91 78.47 76.28 92 107.81 97.16 93 65.74 66.47 9484.06 81.90 95 53.88 67.10 96 97.42 95.63 97 119.93 127.83 98 72.6979.87 99 144.28 143.36 100 149.12 140.94 101 96.03 102.76 102 101.29102.50 103 101.09 108.85 104 109.96 117.58 105 136.29 126.72 106 107.05107.20 107 87.89 91.28 108 143.01 153.30 109 135.15 134.00 110 109.68108.74 111 117.33 123.38 112 128.59 126.45 113 96.53 98.88 114 126.58129.56 115 128.60 131.36 116 117.26 116.58 117 165.65 155.24 118 89.2780.15 119 121.23 125.97 120 129.51 146.03 121 152.74 162.44 122 117.83124.94 123 121.01 125.32 124 122.19 111.80 125 190.94 178.21 126 118.95115.83 127 108.85 110.95 128 76.17 84.41 129 116.38 120.15 130 116.39113.82 131 134.85 142.75 132 173.09 172.40

A comparative linear regression was generated for the data pointscollected in this Example. The linear fit followed the followingequation:Normalized LDL=−0.25+1.00×Serum LDL,

-   -   with the correlation, expressed as R², being equal to 0.96.

EXAMPLE 9

This example demonstrates the measurement of total cholesterol.

Sixty-six patients were used to obtain blood via venal puncture (venousblood specimens) and by pricking their fingers (capillary bloodspecimens). Capillary blood was spotted on xylose-coated Whatman GF/AVAfilter paper, using a device similar to that shown in FIG. 9. Capillaryblood specimens were dried and the portion of the filter paper thatcontained separated serum was cut out and eluted in 8 batches. Eluatefrom each specimen was measured for cholesterol, sodium and chloride onCobas Mira (Roche, Indianapolis, Ind.). The normalized cholesterol levelwas obtained according to the present invention using a formula:Normalized Cholesterol=(Na-Normalized Cholesterol+Cl-NormalizedCholesterol)/2, where Na-Normalized Cholesterol was equal to (MeasuredCholesterol−Cholesterol RD)/(0.0070311+0.8157416×(Fixed SlopeSodium−Sodium Background)/Population Mean Na)) and Cl-NormalizedCholesterol was equal to (Measured Cholesterol−CholesterolRD)/(−0.04833+0.7458044×(Fixed Slope Chloride−ChlorideBackground)/Population Mean CL)). In this equation, A1, B1 and A2, B2were scalar values that were recalculated based on the “tare” procedureheretofore described, whereby a regression for six patients wascalculated and the intercept and slope values from this regression wereused to calculate normalized cholesterol values for specimens.Cholesterol RD was equal to 0.10 mg/dL. Fixed Slope Sodium was equal toMeasured Sodium (or Na)×10 exponent [Log 10 (Na/150)×(Na Slope−Na FixedSlope)/Na Fixed Slope], where Na Fixed Slope=55.7. Fixed Slope Chloridewas equal to Measured Chloride (or Cl)×10 exponent [Log 10 (Cl/138)×(ClSlope−Cl Fixed Slope)/Cl Fixed Slope], where Cl Fixed Slope=55.28.Sodium and Chloride background was equal to 13.32 mEq/L and 12.2 mEq/L.Actual (directly measured in venous blood) and calculated normalizedcholesterol values for these patients are given below.

Na- Cl- Measured Norm Norm Serum Measured Measured Bg- Bg- Na Cl SampleCHO CHO CHO MSS_CHO CHO NA CL Na CL Slope Slope 201 208 253 249 251 268154 135 13.32 12.20 57.37 −39.18 205 231 172 170 171 175 241 249 13.3212.20 57.37 −39.18 206 155 189 188 188 191 153 134 13.32 12.20 57.37−39.18 207 311 277 272 275 288 204 201 13.32 12.20 57.37 −39.18 208 164164 163 163 167 184 171 13.32 12.20 57.37 −39.18 210 145 266 258 262 256108 85 13.32 12.20 57.37 −39.18 211 190 271 271 271 258 133 110 13.3212.20 57.37 −39.18 212 108 201 200 201 185 106 81 13.32 12.20 57.37−39.18 213 188 195 199 197 206 178 158 13.32 12.20 57.37 −39.18 214 134139 138 138 153 179 163 13.32 12.20 57.37 −39.18 215 218 206 209 208 198193 179 13.32 12.20 57.37 −39.18 216 130 247 244 245 254 105 81 13.3212.20 60.07 −40.37 217 221 215 216 216 214 187 173 13.32 12.20 60.07−40.37 219 138 203 204 203 221 131 106 13.32 12.20 60.07 −40.37 220 228199 197 198 211 205 202 13.32 12.20 60.07 −40.37 223 138 195 194 194 193136 113 13.32 12.20 60.07 −40.37 227 283 243 246 245 261 208 199 13.3212.20 60.07 −40.37 230 79 154 157 156 143 103 74 13.32 12.20 58.56−38.21 233 89 174 189 182 170 103 69 13.32 12.20 58.56 −38.21 234 135202 210 206 196 129 99 13.32 12.20 58.56 −38.21 236 103 135 139 137 134144 118 13.32 12.20 58.56 −38.21 237 115 192 199 195 200 118 88 13.3212.20 58.56 −38.21 238 154 192 199 196 204 151 124 13.32 12.20 58.56−38.21 239 77 111 114 112 117 132 104 13.32 12.20 58.56 −38.21 240 95155 143 149 165 119 102 13.32 12.20 58.12 −38.21 241 115 178 183 180 197124 96 13.32 12.20 58.12 −38.21 242 104 190 195 193 211 108 79 13.3212.20 58.12 −38.21 243 107 209 211 210 227 102 75 13.32 12.20 58.12−38.21 244 104 185 189 187 194 110 82 13.32 12.20 58.12 −38.21 245 153176 174 175 178 162 144 13.32 12.20 58.12 −38.21 246 248 312 320 316 314150 124 13.32 12.20 58.12 −38.21 247 137 229 230 230 233 117 90 13.3212.20 58.12 −38.21 248 109 207 209 208 210 104 77 13.32 12.20 58.12−38.21 250 80 138 139 138 144 113 86 13.32 12.20 58.12 −38.21 254 184193 193 193 192 176 161 13.32 12.20 58.12 −38.21 255 145 168 163 166 167161 147 13.32 12.20 57.69 −38.32 256 108 168 159 164 172 124 105 13.3212.20 57.69 −38.32 257 101 192 192 192 188 104 78 13.32 12.20 57.69−38.32 258 223 305 308 307 308 138 114 13.32 12.20 57.69 −38.32 261 310300 296 298 291 189 181 13.32 12.20 57.69 −38.32 262 228 171 169 170 172239 251 13.32 12.20 57.69 −38.32 263 214 190 190 190 192 204 197 13.3212.20 57.69 −38.32 264 256 238 237 237 240 196 188 13.32 12.20 57.69−38.32 265 232 205 205 205 201 205 199 13.32 12.20 57.69 −38.32 266 328296 293 295 302 201 196 13.32 12.20 57.69 −38.32 267 177 208 205 207 198159 141 13.32 12.20 58.02 −42.32 270 137 188 182 185 191 138 120 13.3212.20 58.02 −42.32 271 356 285 275 280 289 225 226 13.32 12.20 58.02−42.32 274 178 185 184 185 193 177 161 13.32 12.20 58.02 −42.32 275 267226 226 226 233 213 202 13.32 12.20 58.02 −42.32 276 235 210 214 212 207203 187 13.32 12.20 58.02 −42.32 277 286 227 226 227 229 225 220 13.3212.20 58.02 −42.32 278 154 170 169 169 172 168 149 13.32 12.20 58.02−42.32 279 194 153 146 149 151 227 233 13.32 12.20 58.02 −42.32 280 155302 303 302 282 102 80 13.32 12.20 58.02 −42.32 283 99 184 178 181 189106 87 13.32 12.20 58.02 −42.32 284 144 192 183 188 188 142 127 13.3212.20 58.02 −42.32 301 101 173 170 171 179 114 95 13.32 12.20 57.04−43.30 302 124 180 178 179 170 131 112 13.32 12.20 57.04 −43.30 305 197220 217 219 229 166 150 13.32 12.20 57.04 −43.30 306 124 191 190 191 198125 104 13.32 12.20 57.04 −43.30 307 227 247 250 249 235 171 150 13.3212.20 57.04 −43.30 308 220 239 236 238 243 170 153 13.32 12.20 57.04−43.30 310 151 235 235 235 237 124 102 13.32 12.20 57.04 −43.30 311 136247 241 244 234 108 90 13.32 12.20 57.04 −43.30 313 169 234 236 235 240137 115 13.32 12.20 57.04 −43.30

A comparative linear regression was generated for the data pointscollected in this Example. The linear fit followed the followingequation:Normalized Cholesterol (MSS CHO)=−1.7+0.996×Serum Cholesterol, with thecorrelation coefficient, expressed as R², being 0. 0.960.

EXAMPLE 10

This example demonstrates the performance of the invention in themeasurement of HDL. The dried spots and venous blood specimens from thesame sixty-six patients in Example 9 were used to measure HDL incapillary blood and compare it to a value for HDL in venous blood. Thenormalized HDL level in capillary blood was obtained according to thepresent invention using the following formula: NormalizedHDL=(Na-Normalized HDL+Cl-Normalized HDL)/2, where Na-Normalized HDL wasequal to (Measured HDL−HDL RD)/(0.0326271+0.8120791×(Fixed SlopeSodium−Sodium Background)/Population Mean Na)) and Cl-Normalized HDL wasequal to (Measured HDL−HDL RD)/(−0.021347+0.7416995×(Fixed SlopeChloride−Chloride Background)/Population Mean CL)), where intercept Aand slope B were obtained as previously described. HDL RD was calculatedas 0.0342×Measured Cholesterol and Sodium and Chloride Background wasthe same as in the Example 9. The following results were observed.

Na- Cl- Measured Norm Norm Serum Measured Measured HDL- Bg- Bg- Na ClSample HDL HDL HDL MSS_HDL HDL NA CL RD Na CL Slope Slope 201 54 55 5555 58 154 135 7.10 13.32 12.20 57.37 −39.18 205 81 54 53 53 54 241 2497.90 13.32 12.20 57.37 −39.18 206 39 40 40 40 40 153 134 5.29 13.3212.20 57.37 −39.18 207 50 35 34 35 37 204 201 10.65 13.32 12.20 57.37−39.18 208 68 61 61 61 62 184 171 5.60 13.32 12.20 57.37 −39.18 210 62100 97 99 91 108 85 4.97 13.32 12.20 57.37 −39.18 211 42 50 50 50 48 133110 6.49 13.32 12.20 57.37 −39.18 212 41 67 67 67 60 106 81 3.70 13.3212.20 57.37 −39.18 213 55 49 50 50 48 178 158 6.45 13.32 12.20 57.37−39.18 214 57 53 53 53 58 179 163 4.59 13.32 12.20 57.37 −39.18 215 8169 69 69 65 193 179 7.46 13.32 12.20 57.37 −39.18 216 32 51 50 51 55 10581 4.43 13.32 12.20 60.07 −40.37 217 69 59 59 59 57 187 173 7.55 13.3212.20 60.07 −40.37 219 35 44 44 44 47 131 106 4.71 13.32 12.20 60.07−40.37 220 65 49 48 49 49 205 202 7.79 13.32 12.20 60.07 −40.37 223 3542 42 42 41 136 113 4.72 13.32 12.20 60.07 −40.37 227 73 53 54 54 50 208199 9.67 13.32 12.20 60.07 −40.37 230 33 56 57 57 53 103 74 2.71 13.3212.20 58.56 −38.21 233 27 45 48 46 45 103 69 3.05 13.32 12.20 58.56−38.21 234 81 111 115 113 108 129 99 4.63 13.32 12.20 58.56 −38.21 23639 45 46 45 40 144 118 3.53 13.32 12.20 58.56 −38.21 237 44 65 67 66 66118 88 3.95 13.32 12.20 58.56 −38.21 238 36 37 39 38 39 151 124 5.2813.32 12.20 58.56 −38.21 239 29 38 39 38 37 132 104 2.62 13.32 12.2058.56 −38.21 240 31 44 41 43 44 119 102 3.24 13.32 12.20 58.12 −38.21241 35 46 47 47 47 124 96 3.92 13.32 12.20 58.12 −38.21 242 34 54 55 5558 108 79 3.55 13.32 12.20 58.12 −38.21 243 21 32 32 32 30 102 75 3.6713.32 12.20 58.12 −38.21 244 22 32 33 32 36 110 82 3.54 13.32 12.2058.12 −38.21 245 58 59 59 59 57 162 144 5.22 13.32 12.20 58.12 −38.21246 76 83 85 84 83 150 124 8.49 13.32 12.20 58.12 −38.21 247 32 44 45 4547 117 90 4.70 13.32 12.20 58.12 −38.21 248 24 36 36 36 34 104 77 3.7113.32 12.20 58.12 −38.21 250 35 53 53 53 61 113 86 2.73 13.32 12.2058.12 −38.21 254 56 51 51 51 46 176 161 6.30 13.32 12.20 58.12 −38.21255 59 61 59 60 56 161 147 4.98 13.32 12.20 57.69 −38.32 256 55 77 73 7570 124 105 3.68 13.32 12.20 57.69 −38.32 257 54 92 92 92 91 104 78 3.4613.32 12.20 57.69 −38.32 258 35 37 37 37 40 138 114 7.62 13.32 12.2057.69 −38.32 261 93 79 78 78 79 189 181 10.61 13.32 12.20 57.69 −38.32262 79 53 52 52 53 239 251 7.80 13.32 12.20 57.69 −38.32 263 68 53 53 5355 204 197 7.31 13.32 12.20 57.69 −38.32 264 91 75 75 75 75 196 188 8.7513.32 12.20 57.69 −38.32 265 90 71 71 71 69 205 199 7.93 13.32 12.2057.69 −38.32 266 64 47 46 46 43 201 196 11.21 13.32 12.20 57.69 −38.32267 51 51 51 51 46 159 141 6.07 13.32 12.20 58.02 −42.32 270 39 46 44 4541 138 120 4.67 13.32 12.20 58.02 −42.32 271 89 61 59 60 60 225 22612.18 13.32 12.20 58.02 −42.32 274 45 40 40 40 40 177 161 6.10 13.3212.20 58.02 −42.32 275 82 61 61 61 61 213 202 9.14 13.32 12.20 58.02−42.32 276 59 44 45 45 42 203 187 8.05 13.32 12.20 58.02 −42.32 277 4930 30 30 31 225 220 9.77 13.32 12.20 58.02 −42.32 278 50 48 48 48 49 168149 5.26 13.32 12.20 58.02 −42.32 279 63 44 42 43 43 227 233 6.62 13.3212.20 58.02 −42.32 280 39 63 63 63 54 102 80 5.31 13.32 12.20 58.02−42.32 283 25 39 38 39 42 106 87 3.38 13.32 12.20 58.02 −42.32 284 51 5957 58 57 142 127 4.91 13.32 12.20 58.02 −42.32 301 29 43 42 42 45 114 953.46 13.32 12.20 57.04 −43.30 302 41 52 51 52 51 131 112 4.23 13.3212.20 57.04 −43.30 305 47 44 43 43 48 166 150 6.75 13.32 12.20 57.04−43.30 306 32 41 41 41 37 125 104 4.25 13.32 12.20 57.04 −43.30 307 5146 47 47 44 171 150 7.78 13.32 12.20 57.04 −43.30 308 72 68 68 68 69 170153 7.52 13.32 12.20 57.04 −43.30 310 37 48 48 48 45 124 102 5.17 13.3212.20 57.04 −43.30 311 22 30 29 30 30 108 90 4.67 13.32 12.20 57.04−43.30 313 53 64 64 64 63 137 115 5.77 13.32 12.20 57.04 −43.30

A comparative linear regression was generated for the data pointscollected in this Example. The linear fit followed the followingequation:Normalized HDL=−1.2+1.035×Serum HDL,with the correlation coefficient, expressed as R², being greater than0.961.

EXAMPLE 11

This example demonstrates the performance of the invention in themeasurement of triglycerides. The dried spots and venous blood specimensfrom the same sixty-six patients in Example 9 were used to measuretriglycerides in capillary blood and compare it to a value fortriglycerides in venous blood. The normalized triglycerides level incapillary blood was obtained according to the present invention using aformula: Normalized Triglycerides=(Na-NormalizedTriglycerides+Cl-Normalized Triglycerides)/2, where Na-NormalizedTriglycerides was equal to (Measured Triglycerides−TriglyceridesRD)/(0.0814243+0.5536861×(Fixed Slope Sodium−SodiumBackground)/Population Mean Na)) and Cl-Normalized Triglycerides wasequal to (Measured Triglycerides−TriglyceridesRD)/(0.0419903+0.5074508×(Fixed Slope Chloride−ChlorideBackground)/Population Mean CL)). Normalized Triglycerides=(MeasuredTriglycerides−Triglycerides RD)/(A+B×(Measured Sodium/139)), where A andB were obtained as previously described. Triglycerides RD was equal to−4.51 mg/dL and Sodium and Chloride Background was the same as in theExample 9.

The following results were observed.

Na- Na- Cl- Measured Norm Norm Norm Serum Measured Measured TG Bg- Bg-Na Cl Sample TG CHO TG TG MSS_TG TG NA CL RD Na CL Slope Slope 201 45253 78 77 78 83 154 135 −4.51 13.32 13.32 57.37 −39.18 205 79 172 84 8384 93 241 249 −4.51 13.32 12.20 57.37 −39.18 206 42 189 74 73 73 78 153134 −4.51 13.32 12.20 57.37 −39.18 207 160 277 196 193 195 208 204 201−4.51 13.32 12.20 57.37 −39.18 208 21 164 34 34 34 35 184 171 −4.5113.32 12.20 57.37 −39.18 210 23 266 62 60 61 66 108 85 −4.51 13.32 12.2057.37 −39.18 211 107 271 202 202 202 196 133 110 −4.51 13.32 12.20 57.37−39.18 212 90 201 215 215 215 197 106 81 −4.51 13.32 12.20 57.37 −39.18213 156 195 219 223 221 198 178 158 −4.51 13.32 12.20 57.37 −39.18 21469 139 100 100 100 85 179 163 −4.51 13.32 12.20 57.37 −39.18 215 67 20690 91 90 88 193 179 −4.51 13.32 12.20 57.37 −39.18 216 103 247 250 247248 201 105 81 −4.51 13.32 12.20 60.07 −40.37 217 91 215 123 124 124 105187 173 −4.51 13.32 12.20 60.07 −40.37 219 102 203 199 200 200 156 131106 −4.51 13.32 12.20 60.07 −40.37 220 75 199 93 92 92 82 205 202 −4.5113.32 12.20 60.07 −40.37 223 73 195 140 139 139 133 136 113 −4.51 13.3212.20 60.07 −40.37 227 184 243 217 220 218 205 208 199 −4.51 13.32 12.2060.07 −40.37 230 20 154 57 58 57 51 103 74 −4.51 13.32 12.20 58.56−38.21 233 38 174 101 109 105 96 103 69 −4.51 13.32 12.20 58.56 −38.21234 49 202 101 104 102 84 129 99 −4.51 13.32 12.20 58.56 −38.21 236 73135 130 133 132 118 144 118 −4.51 13.32 12.20 58.56 −38.21 237 28 192 6769 68 58 118 88 −4.51 13.32 12.20 58.56 −38.21 238 42 192 76 78 77 82151 124 −4.51 13.32 12.20 58.56 −38.21 239 35 111 72 74 73 57 132 104−4.51 13.32 12.20 58.56 −38.21 240 50 155 112 105 108 98 119 102 −4.5113.32 12.20 58.12 −38.21 241 20 178 47 48 48 56 124 96 −4.51 13.32 12.2058.12 −38.21 242 57 190 138 142 140 144 108 79 −4.51 13.32 12.20 58.12−38.21 243 101 209 248 250 249 268 102 75 −4.51 13.32 12.20 58.12 −38.21244 153 185 345 351 348 424 110 82 −4.51 13.32 12.20 58.12 −38.21 245 45176 74 74 74 66 162 144 −4.51 13.32 12.20 58.12 −38.21 246 39 312 71 7372 77 150 124 −4.51 13.32 12.20 58.12 −38.21 247 39 229 90 91 91 93 11790 −4.51 13.32 12.20 58.12 −38.21 248 49 207 124 125 124 132 104 77−4.51 13.32 12.20 58.12 −38.21 250 23 138 58 59 58 47 113 86 −4.51 13.3212.20 58.12 −38.21 254 59 193 87 87 87 84 176 161 −4.51 13.32 12.2058.12 −38.21 255 132 168 205 200 203 190 161 147 −4.51 13.32 12.20 57.69−38.32 256 23 168 53 51 52 47 124 105 −4.51 13.32 12.20 57.69 −38.32 25725 192 68 68 68 66 104 78 −4.51 13.32 12.20 57.69 −38.32 258 89 305 164165 164 165 138 114 −4.51 13.32 12.20 57.69 −38.32 261 80 300 108 107107 107 189 181 −4.51 13.32 12.20 57.69 −38.32 262 48 171 53 53 53 59239 251 −4.51 13.32 12.20 57.69 −38.32 263 71 190 90 91 91 93 204 197−4.51 13.32 12.20 57.69 −38.32 264 45 238 61 61 61 69 196 188 −4.5113.32 12.20 57.69 −38.32 265 40 205 53 53 53 55 205 199 −4.51 13.3212.20 57.69 −38.32 266 395 296 482 478 480 501 201 196 −4.51 13.32 12.2057.69 −38.32 267 133 208 211 208 209 205 159 141 −4.51 13.32 12.20 58.02−42.32 270 121 188 221 215 218 217 138 120 −4.51 13.32 12.20 58.02−42.32 271 111 285 124 121 122 127 225 226 −4.51 13.32 12.20 58.02−42.32 274 75 185 108 108 108 115 177 161 −4.51 13.32 12.20 58.02 −42.32275 83 226 99 99 99 109 213 202 −4.51 13.32 12.20 58.02 −42.32 276 177210 217 220 218 208 203 187 −4.51 13.32 12.20 58.02 −42.32 277 300 227328 325 326 309 225 220 −4.51 13.32 12.20 58.02 −42.32 278 43 170 69 6969 73 168 149 −4.51 13.32 12.20 58.02 −42.32 279 60 153 68 66 67 76 227233 −4.51 13.32 12.20 58.02 −42.32 280 74 302 186 186 186 184 102 80−4.51 13.32 12.20 58.02 −42.32 283 24 184 65 63 64 68 106 87 −4.51 13.3212.20 58.02 −42.32 284 62 192 115 110 112 106 142 127 −4.51 13.32 12.2058.02 −42.32 301 32 173 78 77 77 75 114 95 −4.51 13.32 12.20 57.04−43.30 302 59 180 116 115 116 95 131 112 −4.51 13.32 12.20 57.04 −43.30305 92 220 142 140 141 158 166 150 −4.51 13.32 12.20 57.04 −43.30 306 35191 77 77 77 84 125 104 −4.51 13.32 12.20 57.04 −43.30 307 144 247 211214 212 201 171 150 −4.51 13.32 12.20 57.04 −43.30 308 71 239 109 107108 111 170 153 −4.51 13.32 12.20 57.04 −43.30 310 84 235 172 172 172174 124 102 −4.51 13.32 12.20 57.04 −43.30 311 438 247 982 962 972 989108 90 −4.51 13.32 12.20 57.04 −43.30 313 43 234 84 85 85 87 137 115−4.51 13.32 12.20 57.04 −43.30

A comparative linear regression was generated for the data pointscollected in this Example. The linear fit followed the followingequation:Normalized TG=6.4+0.967×Serum TG,

-   -   with the coefficient, expressed as R², being 0.987.

EXAMPLE 12

This example demonstrates the performance of the invention in themeasurement of LDL. The same observations from the same six patients inExample 9, 10 and 11 were used to calculate a value for LDL in serum anda value for LDL in MSS according to the Friedewald formula:Serum LDL=Serum Cholesterol−Serum HDL−Serum TG/5Normalized LDL=Normalized Cholesterol−Normalized HDL−Normalized TG/5.LDL was not calculated when Triglycerides were above 400 mg/dL.

The following results were calculated:

Serum Sample MSS_CHO MSS_HDL MSS_TG MSS_LDL LDL 201 251 55 78 181 194205 171 53 84 101 102 206 188 40 73 134 136 207 275 35 195 201 210 208163 61 34 95 98 210 262 99 61 151 152 211 271 50 202 181 171 212 201 67215 91 86 213 197 50 221 103 118 214 138 53 100 65 78 215 208 69 90 121115 216 245 51 248 145 159 217 216 59 124 132 137 219 203 44 200 120 142220 198 49 92 131 146 223 194 42 139 125 125 227 245 54 218 147 170 230156 57 57 88 80 233 182 46 105 114 106 234 206 113 102 73 71 236 137 45132 65 70 237 195 66 68 116 123 238 196 38 77 142 149 239 112 38 73 5968 240 149 43 108 85 101 241 180 47 48 124 139 242 193 55 140 110 124243 210 32 249 128 143 244 187 32 348 85 74 245 175 59 74 101 108 246316 84 72 218 216 247 230 45 91 167 167 248 208 36 124 147 150 250 13853 58 73 74 254 193 51 87 124 129 255 166 60 203 65 73 256 164 75 52 7892 257 192 92 68 86 84 258 307 37 164 237 235 261 298 78 107 199 191 262170 52 53 107 107 263 190 53 91 119 119 264 237 75 61 150 152 265 205 7153 124 120 266 295 46 480 267 207 51 209 114 110 270 185 45 218 97 106271 280 60 122 196 203 274 185 40 108 123 129 275 226 61 99 146 150 276212 45 218 123 123 277 227 30 326 131 136 278 169 48 69 107 108 279 14943 67 93 93 280 302 63 186 202 192 283 181 39 64 130 134 284 188 58 112107 109 301 171 42 77 113 119 302 179 52 116 104 100 305 219 43 141 147150 306 191 41 77 134 144 307 249 47 212 159 151 308 238 68 108 148 151310 235 48 172 153 157 311 244 30 972 313 235 64 85 154 160

A comparative linear regression was generated for the data pointscollected in this Example. The linear fit followed the followingequation:Normalized LDL=−2.49+0.99×Serum LDL,with the correlation, expressed. as R², being equal to 0.960.

It is thus seen that the invention provides a method for determining thelevel of an analyte in a specimen.

While particular embodiments to the invention have been describedherein, the invention is not limited thereto, but to the contrary shouldbe deemed defined by the full scope of the appended claims. Allreferences and prior and co-pending applications cited herein are herebyincorporated by reference in their entireties.

1. A method for determining the level of an analyte in blood from asolution formed from a dried blood fluid specimen, said blood fluidspecimen being a plasma or serum specimen, comprising: in either order,measuring the analyte level in said solution and measuring the level ofat least one normalizing analyte; and determining the analyte level inthe blood from which said blood fluid specimen was collected based onsaid analyte level in said solution and on the level of said normalizinganalyte in said solution, wherein, in determining the analyte level inthe blood from which said blood fluid specimen was collected, a recoverydelta is added to the analyte level of the analyte level in saidsolution to provide a recovery delta corrected solution analyte level,and the analyte level in the blood from which said blood fluid specimenwas collected is determined as a function of said recovery deltacorrected solution analyte level and on the level of said normalizinganalyte, wherein said recovery delta is a positive value, wherein saidrecovery delta is determined as a function of the time elapsed sincesaid blood fluid specimen was collected.
 2. A method according to claim1, wherein said recovery delta is determined in accordance with thefunction: A+B (time) wherein A is 0, a positive value, or a negativevalue, and B is a positive or negative non-zero value.
 3. A methodaccording to claim 2, wherein B is determined based on climacticconditions.
 4. A method according to claim 1, said analyte beingselected from the group consisting of total cholesterol, HDL, LDL, andTG.
 5. A method according to claim 1, said normalizing analyte includingsodium.
 6. A method according to claim 1, said normalizing analyteincluding chloride.
 7. A method for determining the level of an analytein blood from a solution formed from a dried blood fluid specimen, saidblood fluid specimen being a plasma or serum specimen, comprising: ineither order, measuring the analyte level in said solution and measuringthe level of at least one normalizing analyte; and determining theanalyte level in the blood from which said blood fluid specimen wascollected based on said analyte level in said solution and on the levelof said normalizing analyte in said solution, wherein, in determiningthe analyte level in the blood from which said blood fluid specimen wascollected, a recovery delta is added to the analyte level of the analytelevel in said solution to provide a recovery delta corrected solutionanalyte level, and the analyte level in the blood from which said bloodfluid specimen was collected is determined as a function of saidrecovery delta corrected solution analyte level and on the level of saidnormalizing analyte, wherein said recovery delta is a negative value,wherein said recovery delta is determined as a function of the timeelapsed since said blood fluid specimen was collected.
 8. A method fordetermining the level of an analyte in blood from a solution formed froma dried blood fluid specimen, said blood fluid specimen being a plasmaor serum specimen, comprising: in either order, measuring the analytelevel in said solution and measuring the level of at least a firstnormalizing analyte; and determining an analyte level in the blood fromwhich said blood fluid specimen was collected based on said analytelevel in said solution and on the level of said first normalizinganalyte in said solution, wherein the calculation of analyte level inthe blood from which said blood fluid specimen was taken includes apredetermined nonlinear first analyte correction factor, measuring thelevel of at least a second normalizing analyte in said solution, whereinthe level of analyte in the blood from which said blood fluid specimenwas collected is determined based on the level of said secondnormalizing analyte and on the corrected first analyte level.
 9. Amethod according to claim 8, wherein a first blood analyte level isdetermined based on the levels in solution of said analyte and saidchloride normalizing analyte; a second blood analyte level is determinedbased on the levels in solution of said analyte and said secondnormalizing analyte; and the level of analyte in the blood from whichsaid blood fluid specimen was collected is determined by calculating themean average of said first and second blood analyte levels.
 10. A methodaccording to claim 9, said first normalizing analyte comprisingchloride.
 11. A method for reporting a test result, comprising:receiving an incoming inquiry from a user; prompting said user for atest number; retrieving a test result from a database of test numbersand test results; and reporting said test result to said user, said testresult comprising an analyte level having been determined according to amethod for determining the level of an analyte in blood from a solutionformed from a dried blood fluid specimen, said blood fluid specimenbeing a plasma or serum specimen, comprising: in either order, measuringthe analyte level in said solution and measuring the level of at leastone normalizing analyte; and determining the analyte level in the bloodfrom which said blood fluid specimen was collected based on said analytelevel in said solution and on the level of said normalizing analyte insaid solution, wherein, in determining the analyte level in the bloodfrom which said blood fluid specimen was collected, a recovery delta isadded to the analyte level of the analyte level in said solution toprovide a recovery delta corrected solution analyte level, and theanalyte level in the blood from which said blood fluid specimen wascollected is determined as a function of said recovery delta correctedsolution analyte level and on the level of said normalizing analyte. 12.A method for reporting a test result, comprising: receiving an incominginquiry from a user; prompting said user for a test number; retrieving atest result from a database of test numbers and test results; andreporting said test result to said user, said test result comprising ananalyte level having been determined according to a method fordetermining the level of an analyte in blood from a solution formed froma dried blood fluid specimen, said blood fluid specimen being a plasmaor serum specimen, comprising: in either order, measuring the analytelevel in said solution and measuring the level of at least a firstnormalizing analyte; and determining the analyte level in the blood fromwhich said blood fluid specimen was collected based on said analytelevel in said solution and on the level of said first normalizinganalyte in said solution, wherein the calculation of analyte level inthe blood from which said blood fluid specimen was taken includes apredetermined nonlinear first analyte correction factor.